Accurate, inexpensive and efficient methods of large scale mutagenesis of specific DNA sequences have the potential to allow for rapid assessment of the effects of single nucleotide changes on protein function. These methods remain limited by the need to synthesize oligonucleotides en masse to be used as templates for mutagenesis. Currently, the construction of allelic series requires the purchase of many hundreds to thousands of oligonucleotides of 10s of bases to hundreds of bases with specific sequences. Thus, there is a need in the art for a new method of single nucleotide mutagenesis where the purchase of these oligos is no longer required to produce allelic series pools.
Such a novel method would allow for the construction of pools of DNA sequences that differ from the normal form by one and only one base allowing for the rapid production of allelic series to be used in structure-function studies. This method would aid in the interpretation of variants observed in human patients of unknown significance and for in vitro protein evolution and engineering.